Newer approaches to the treatment of HIV infection seek to block the entry of the virus into the cell by targeting different proteins engaged in the entry process. Possible targets for entry inhibition include the HIV envelope (Env) glycoproteins gp120 and gp41, the cellular receptor CD4, and the chemokine receptors CCR5 and CXCR4. Clinical trials have demonstrated anti-HIV potential of the entry inhibitors. However, they suffer from the same challenges as the other HAART drugs - rapid outgrowth of resistant virus and numerous side effects. Recently, cell cycle agents, such as rapamycin that downregulates the CCR5 receptor, were found to synergize with the entry inhibitor T20, one of the two entry inhibitors approved by the FDA, known also as Fuzeon or Enfuvirtide. Our preliminary experiments further demonstrate that the addition of rapamycin in vitro prevents the emergence of T20 resistant viruses and enhances the susceptibility to T20 of otherwise resistant viruses. We have also found that rapamycin synergizes with and enhances the anti-HIV efficiency of TAK-779, a small molecule CCR5 antagonist with potent antiviral activity. These observations suggest that rapamycin may enhance the therapeutic performance of other entry inhibitors such as selzentry, known as maraviroc, which, like TAK-779, targets CCR5. Selzentry has already been approved by the FDA on August 6, 2007 for anti-HIV therapy in combinations with other HAART drugs. The objective of this project, therefore, is to identify those small molecule entry inhibitors that are on the market and in advanced clinical or preclinical studies that show improved anti-HIV efficiency in the presence of rapamycin. We propose to reach the goal via the following 3 aims: Aim 1: Identify which of the selected small molecule entry inhibitors synergize with rapamycin to inhibit viral infections in human PBMCs; Aim 2: Demonstrate that the addition of rapamycin inhibits the outgrowth of inhibitor resistant viruses in vitro; Aim 3: Demonstrate that the addition of rapamycin can recover the susceptibility of the inhibitor resistant viruses. Should we identify compounds with the preceding characteristics during this Phase I project, our SBIR Phase II proposal will endeavor to develop an oral rapamycin/entry inhibitor co-formulation for treatment of HIV infection. This document contains proprietary information that Profectus BioSciences requests not be released to persons outside the Government, except for purposes of review and evaluation. PUBLIC HEALTH RELEVANCE Rapamycin (Wyeth, Madison, NJ) is an immunosuppressant drug prescribed for oral use after kidney transplantation. It is a cell cycle agent that downregulates the expression of CCR5 on the surface of activated T cells. In vitro, rapamycin enhances the efficacy of HIV entry inhibitors: T20 and TAK-779. This observation suggests that rapamycin may also enhance the potential of small molecule entry inhibitors that have already shown reasonable bioavailability. The objective of this project is to identify orally bioavailable entry inhibitors that are on the market, near market or in early clinical studies and that synergize with rapamycin in their anti-HIV efficiency. Such inhibitors will provide opportunities to develop novel orally administered formulations with enhanced antiviral efficiency. Preclinical and clinical development of such combinations will be the subject of follow-on SBIRs. [unreadable] [unreadable] [unreadable]